In this study, the selectivity and specificity of aldose reductase (AR) for glutathionyl aldehydes was examined. Relative to free aldehydes, AR was a more efficient catalyst for the reduction of glutathiolated aldehydes. Reduction of glutathionyl propanal [γGlu-Cys(propanal)-Gly] was more efficient than that of Gly-Cys(propanal)-Gly and γ-aminobutyric acid-Cys(propanal)-Gly suggesting a possible interaction between α-carboxyl of the conjugate and AR. Two active site residues, Trp20 or Ser302, were identified by molecular modeling as potential sites of this interaction. Mutations containing tryptophan-to-phenylalanine (W20F) and serine-to-alanine (S302A) substitutions did not significantly affect reduction of free aldehydes but decreased the catalytic efficiency of AR for glutathiolated aldehydes. Combined mutations indicate that both Trp20 and Ser302 are required for efficient catalysis of the conjugates. The decrease in efficiency due to W20F mutation with glutathionyl propanal was not observed with γ-aminobutyric-Cys(propanal)-Gly or Gly-Cys-(propanal)-Gly, indicating that Trp20 is involved in binding the α-carboxyl of the conjugate. The effect of the S302A mutation was less severe when γGlu-Cys-(propanal)-Glu rather than glutathionyl propanal was used as the substrate, consistent with an interaction between Ser302 and Gly-3 of the conjugate. These observations suggest that glutathiolation facilitates aldehyde reduction by AR and enhances the range of aldehydes available to the enzyme. Because the N-terminal carboxylate is unique to glutathione, binding of the conjugate with the α-carboxyl facing the bottom of the α/β-barrel may assist in the exclusion of unrelated peptides and proteins.
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